Eliminate sequence constraints for dsDNA targeting with non-specific PAM (5´-NNN-3´ PAM)
Digest large plasmids in cloning workflows successfully
Use in conjunction with the EnGen sgRNA Synthesis Kit, S. pyogenes (NEB #E3322), EnGen Mutation Detection Kit (NEB #E3321), and NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621)
EnGen SpRY Cas9 from Streptococcus pyogenes is an engineered, RNA-guided, DNA endonuclease that catalyzes site-specific cleavage of double-stranded DNA (dsDNA). Targeting requires a ~100 nucleotide single guide RNA (sgRNA) with complementarity to the 20-nucleotide region immediately upstream of a protospacer adjacent motif (PAM) on the dsDNA substrate. EnGen SpRY Cas9 encodes 11 point mutations (A61R, L1111R, D1135L, S1136W, G1218K, E1219Q, N1317R, A1322R, R1333P, R1335Q, T1337R) designed to diminish the requirement for a PAM. Unlike the canonical 5´-NGG-3´ PAM requirement of wild-type Spy Cas9, SpRY Cas9 has been demonstrated to have almost no PAM requirement in vitro, cleaving at many sites with a 5´-NNN-3´ PAM (although it exhibits a preference for 5´-NRN-3´ over 5´-NYN-3´ PAMs in vivo) (1,2). DNA cleavage by EnGen SpRY Cas9 produces a double-stranded break occurring 3 nucleotides upstream of the PAM. EnGen SpRY Cas9 contains Simian virus 40 (SV40) T antigen nuclear localization sequence (NLS) on the C-terminus of the protein.
Figure 1: EnGen SpRY Cas9 has no sequence constraints for dsDNA targeting in vitro
EnGen SpRY Cas9 is a variant of Cas9 nuclease from S. pyogenes with several point mutations within the PAM interacting domain (1). Unlike wildtype Cas9, EnGen SpRY Cas9 is not constrained by the presence of an NGG PAM and can produce a double stranded break three nucleotides upstream of any trinucleotide sequence in in vitro applications.
Figure 2: Streamline large construct cloning workflows with EnGen SpRY Cas9 and NEBuilder HiFi DNA Assembly
Demonstration of EnGen® SpRY Cas9 cleavage flexibility. A) Schematic of pXba with restriction endonuclease BsrGI recognition sites denoted. The rectangular box contains the BsrGI recognition sequence with the cleavage sites denoted by red triangles. B) Sequences of three sgRNAs used to target BsrGI sites in pXba. Cleavage sites for both EnGen SpRY Cas9 and BsrGI are denoted by red triangles. SpRY Cas9 non-canonical PAMs are denoted in yellow text. C) 1 µg of pXba (22,563 bp) was digested with either 10 units of BsrGI or 1 µM EnGen SpRY Cas9 and 1 µM each of three sgRNAs targeting all three BsrGI sites present in pXba in 1X NEBuffer r3.1. All reactions were incubated at 37°C for 1 hour followed by 80°C for 5 minutes. The DNA banding pattern of the BsrGI and SpRY digests were compared by gel electrophoresis using the Agilent gDNA ScreenTape System on a 4200 TapeStation instrument. Cleavage of pXba by both BsrGI and SpRY Cas9 targeted to the BsrGI sites resulted in a nearly identical band pattern. Digestion of 1 µg of pXba with EnGen SpRY Cas9 and sgRNA concentrations as low as 50 nM resulted in a similar cleavage compared with 1 µM results. Undigested pXba was run as a control, however circular DNA does not run accurate to size in the gDNA ScreenTape System. A vertical grey line marks where two different ScreenTapes were used.
Figure 3: Linearization of a 22.6 kb plasmid with EnGen SpRY Cas9 followed by cloning with NEBuilder HiFi DNA Assembly.
Determination of suitability of EnGen SpRY Cas9 digestion reactions for subsequent use in NEBuilder HiFi DNA Assembly reactions. A) Schematic depicting the linearization and cloning strategy. 1 µg of pXba (22,563 bp) was linearized downstream of the start codon of the pVII orf with 50 nM EnGen SpRY Cas9 and 50 nM sgRNA (targeting sequence of 5´-CTTTTTGAGCAAACATGTCC-3´) in 1X NEBuffer r3.1 for 1 hour at 37°C. The reactions were either spin column purified or left unpurified before proceeding to DNA assembly. The NEBuilder HiFi DNA Assembly kit was used to insert an oligonucleotide encoding a nuclear localization signal (NLS) tag to pVII according to the recommended protocol. B) Inspection of purified and unpurified linearized pXba after EnGen SpRY Cas9 digestion using the Agilent gDNA ScreenTape System. Undigested pXba was run as a control, however circular DNA does not run accurate to size in the gDNA ScreenTape System. C) Measurement of the number of colonies grown after transformation with DNA assembly reactions using either purified or unpurified EnGen SpRY Cas9 digests. A no insert control was included to qualitatively assess how many transformants arise from undigested plasmid following EnGen SpRY Cas9 digest. Purification of linearized pXba prior to DNA assembly reduced the percentage of background colonies. 8-9 colonies of transformants derived from purified and unpurified EnGen SpRY Cas9 digests were picked and checked for proper DNA assembly by PCR using primers spanning the insert. 100 percent of clones from both purified and unpurified EnGen SpRY Cas9 digests displayed the correct insert (data not shown).
Product Source
An E. coli strain that carries the cloned Cas9 gene from Streptococcus pyogenes with C-terminal Simian virus 40 (SV40) T antigen nuclear localization signal (NLS) and a C-terminal 6XHis tag
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